The advent of hybridoma technology, originally described by Kohler and Milstein (Nature 256: 495, 1975), has made possible the production of antibodies specific to antigens of various cells. Briefly, the production of monoclonal antibodies involves essentially three steps: (a) imminizing a rodent with either intact tumor cells or cell fractions; (b) fusing myeloma cells with spleen cells obtained from the immunized animal in order to produce hybridomas secreting the desired antibody; and (c) culturing the hybridomas and selecting the desired hybridoma clone.
However, not all of the hybridoma clones which result from the procedure described above are directed at the desired antigen. If one utilizes whole tumor cells to induce antibodies to tumor-associated antigens, typically many antibodies are produced to highly immunogenic, non-tumor associated molecules such as histocompatibility antigens or peripheral proteins like fibronectin. If one attempts to reduce the complexity of the immunogen by procedures to enrich or purify certain tumor associated molecules, the immunogenicity of the soluble form of antigen is poor, even when using immunization protocols known to give high-titered antisera in rabbits or goats. Rodents typically require an antigen in an insoluble form. Thus, the number of specifically reactive hybrids obtained is usually low.
Several attempts have been made to narrow the range of monoclonal antibodies elicited. For example, Middleton et al. (Fed. Proc. 39: 3464, 1980) disclosed a method of inducing tolerance to B lymphocytes in order to enhance the percentage of T-cell-specific hybridomas. Kennett et al. (Top. Micro. Immunol. 81: 77, 1978) describe a method for increasing specificity by blocking surface antigens of one type of cell with whole antiserum to another type of cell not bearing the antigen desired. More recently, U.S. Pat. No. 4,427,653 discloses a method in which antigen mixtures are adsorbed with one or more previously isolated monoclonal antibodies to remove competing immunogenic antigens. Thus, immunogenicity of residual antigens is enhanced due to reduced competition from the depleted antigens.
These methods of immunization are not entirely satisfactory, because either the efficiency of inducing an antibody of choice is low, or because the method requires purified antigen which is difficult to isolate, or requires preexisting monoclonals or polyclonal antisera.
Within the current state of the art, there is particular interest in methods for improving the efficiency of elicitation of monoclonal antibodies to human tumorassociated antigens (TAA). Most of the research efforts have involved the use of intact tumor cells for both the elicitation and evaluation of hybridoma products.
Also of interedst are methods of inducing monoclonal antibodies of the IgG class, in particular the IgG.sub.3 subclass. Recent reports with the IgG.sub.3 monoclonal antibody to the glycolipid antigen GD.sub.3 have indicated that monoclonal antibodies mediating antibody-dependent cellular cytotoxicity (ADCC) in vitro may also mediate anti-tumor effects in patients receiving these unconjugated monoclonal antibodies (Cardon-Cardo et al., Fed. Proc. 44: 8483, 1985). In contrast, monoclonals of other subclasses do not mediate ADCC as effectively. Monoclonal antibodies of the IgG.sub.3 subclass are, however, typically very rare in fusions generated from whole-cell immunizations.
There exists a need in the art, then, for methods of inducing the production of IgG-class monoclonal antibodies, in particular the IgG.sub.3 subclass, as well as a method for improving the efficiency of elicitation of monoclonal antibodies to glycoproteins and, in particular, tumor-associated antigens. The present invention fulfills these needs and further provides other related advantages.